Differential effects of TRPM4 channel inhibitors on Guinea pig urinary bladder smooth muscle excitability and contractility: Novel 4‐chloro‐2‐[2‐(2‐chloro‐phenoxy)‐acetylamino]‐benzoic acid (CBA) versus classical 9‐phenanthrol

Abstract Non‐selective cation channels in urinary bladder smooth muscle (UBSM) are thought to mediate increases in cellular excitability and contractility. For transient receptor potential melastatin type‐4 (TRPM4) channels, the evidence primarily relies on the inhibitor 9‐phenanthrol, which exhibits pharmacological limitations. Recently, 4‐chloro‐2‐[2‐(2‐chloro‐phenoxy)‐acetylamino]‐benzoic acid (CBA) has been discovered as a novel TRPM4 channel blocker. We examined how, in comparison to 9‐phenanthrol, CBA affects the excitability of freshly isolated guinea pig UBSM cells and the contractility of UBSM strips. Additionally, non‐selective TRPM4 channel inhibitor flufenamic acid (FFA) and potentiator BTP2 (also known as YM‐58483) were studied in UBSM cells. Unlike robust inhibition for 9‐phenanthrol already known, CBA (up to 100 μM) displayed either no or a very weak reduction (<20%) in spontaneous phasic, 20 mM KCl‐induced, and electrical field stimulated contractions. For 300 μM CBA, reductions were higher except for an increase in the frequency of KCl‐induced contractions. In UBSM cells, examined under amphotericin B‐perforated patch‐clamp, CBA (30 μM) did not affect the membrane potential (I = 0) or voltage step‐induced whole‐cell cation currents, sensitive to 9‐phenanthrol. The currents were not inhibited by FFA (100 μM), increased by BTP2 (10 μM), nor enhanced under a strongly depolarizing holding voltage of −16 or + 6 mV (vs. −74 mV). None of the three compounds affected the cell capacitance, unlike 9‐phenanthrol. In summary, the novel inhibitor CBA and nonselective FFA did not mimic the inhibitory properties of 9‐phenanthrol on UBSM function. These results suggest that TRPM4 channels, although expressed in UBSM, play a distinct role rather than direct regulation of excitability and contractility.


| INTRODUC TI ON
Urinary bladder smooth muscle (UBSM) relaxation and contraction determine urine storage or voiding, respectively. 1 UBSM cells express various types of ion channels that either inhibit (e.g., voltagedependent K + channel type 2 or K V 2, K V 7, and K Ca 1.1) or promote (e.g., voltage-gated Ca 2+ channel type 1.2 or Ca V 1.2 and Ca V 3) cell excitability. 2 Seminal studies on guinea pigs 3 and rats 4 and later on humans 5 and mice 6,7 identified the transient receptor potential melastatin type-4 (TRPM4) channel as a likely regulator of UBSM excitation-contraction coupling.
CBA inhibits TRPM4 channels with a 20-fold greater potency than 9-phenanthrol. 20 Other modulators, albeit non-selective, used to study TRPM4 channels are flufenamic acid (FFA) and BTP2 (also known as YM-58483), which, respectively, block and potentiate them. 20,26,27 In this study, we aimed to further corroborate the role of TRPM4 channels in UBSM excitability and contractility using the novel TRPM4 channel inhibitor, CBA, and two other known modulators, the activator BTP2 and inhibitor FFA, in guinea pig UBSM. We found unexpectedly that CBA and FFA did not mimic the inhibitory effect of 9-phenanthrol nor did we observe any change for BTP2 on UBSM excitability. We elaborate on the implications of these novel findings regarding TRPM4 channel function in UBSM. 662 g, 25th percentile: 538 g; 75th percentile: 931 g) were euthanized either with CO 2 or isoflurane (Forane ® , Baxter, Deerfield, IL) followed by thoracotomy. Then, the whole bladder was cut above the bladder neck and transferred to a Petri dish containing dissection/digestion solution (see Solutions and compounds section for composition). The whole bladder was excised, and the mucosa including urothelium removed. UBSM strips (5-10 mm long and 2-4 mm wide) were prepared for isometric tension recordings and for UBSM single-cell isolation.

| Fresh enzymatic single UBSM cell isolation
Guinea pig UBSM cells were isolated as previously described. 3

| Electrophysiological experiments
Amphotericin B-perforated whole-cell patch-clamp recordings were performed based on methods and procedures previously described. 3,15,28 Briefly, several drops of the dissection/digestion solution containing freshly isolated UBSM cells were placed into a recording chamber. After at least 45 min, the cells were washed several times with the extracellular (bath) solution (see Solutions and compounds). Voltage step-induced currents or membrane potentials (current clamp, I = 0) were recorded using an Axopatch

| Isometric UBSM tension recordings
The isometric UBSM tension recording experiments were conducted as previously described. 3,13,15 Briefly, UBSM strips were clipped between a stationary mount and a force-displacement transducer and then placed in tissue baths filled with Ca 2+containing physiological salt solution (PSS) thermostatically controlled at 37°C. UBSM strips were stretched to an initial level of For EFS studies, after the equilibration period, UBSM strips were subjected to continuous stimulation with a 10 Hz stimulation frequency at 1 min intervals and increasing concentrations of CBA were applied cumulatively at 10 to 12 min intervals. Unlike spontaneous and 20 mM KCl-induced phasic contractions, all EFS responses were recorded in the absence of tetrodotoxin (1 μM).

| Solutions and compounds
The dissection/digestion solution with or without supplemented

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy. org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, 31

| Differential effects of CBA and 9-phenanthrol on UBSM spontaneous phasic, 20 mM KCl-induced phasic, and EFS-evoked contractions
We have previously shown that 9-phenanthrol, a classical TRPM4 channel blocker, effectively inhibited guinea pig UBSM contractility 3,15 ; see also Tables 1-3. Here, we further examined how the novel TRPM4 channel inhibitor CBA affects UBSM contractility in the same animal species. The effects of CBA were examined on spontaneous phasic ( Figure 1 and Table 1), 20 mM KCl-induced ( Figure 2 and Table 2), and EFS-evoked contractions ( Figure 3 and  Figure 3 and Table 3).
Of note, the frequency for 20 mM KCl-induced phasic contractions, unlike spontaneous phasic and EFS-evoked contractions, increased at the highest concentrations tested (Figure 2A,B and Table 2). The comparison of responses to CBA and 9-phenanthrol on the three types of contractions revealed that the effects of the former compound were substantially weaker (Tables 1-3). The absence of a robust inhibitory effect of CBA on UBSM contractility was surprising given the compound was reported to have ~20-fold higher potency and comparable maximum efficacy to 9-phenanthrol for TRPM4 channels. 20  To further demonstrate that the specific UBSM cells tested still retained sensitivity to 9-phenanthrol, this widely used inhibitor was subsequently added to the same UBSM cells previously evaluated for FFA following its washout. 9-Phenanthrol effectively attenuated voltage-step-induced cation currents ( Figure 6). In contrast to FFA, 9-

| BTP2, a nonselective TRPM4 channel activator, does not increase 9-phenanthrol-sensitive voltage-step-induced cation currents in isolated UBSM cells
Among the various pharmacological effects of BTP2, enhancement of TRPM4 currents has been reported. 27 Here, the effect of BTP2 (10 μM) was evaluated on the voltage-step-induced cation currents.
BTP2 did not alter the currents (Figure 7). The subsequent addition

| Depolarizing holding potentials do not increase voltage-step-induced cation currents in isolated UBSM cells
A previous study on recombinant and native TRPM4 channels reported that TRPM4 currents display dependency on the holding voltage. The TRPM4 currents show enhancement upon a strong depolarizing holding voltage. 27 Here, the voltage-step-induced currents in UBSM cells were examined at the standard holding voltage

| DISCUSS ION
The role of TRPM4 channels in UBSM function is primarily inter- CBA displays ~3-to 10-fold higher potency than 9-phenanthrol, but comparable maximum efficacy on recombinant TRPM4 currents. 20 We hypothesized that CBA would be more potent than 9-phenanthrol in inhibiting UBSM contractions. Instead, CBA displayed either no or weak inhibition on spontaneous, 20 mM KCl-induced, and EFS-induced contractions (Figures 1-3). The modest CBA-induced inhibition observed at the highest concentrations likely reflects non-selectivity. 36 In contrast, 9-phenanthrol strongly inhibited guinea pig UBSM contractility. 3,13,15 In our experiments, the maximum concentration of DMSO to dissolve CBA was 0.3% (v/v). At this concentration, the vehicle exhibits only modest effects on guinea pig UBSM contractility specifically increases up to 30% on the amplitude and muscle force and no significant effects on other contraction parameters that were associated with ~20% reduction in the tone. 15 The effects of CBA and 9-phenanthrol are, hence, distinct from the vehicle. CBA failed to alter the membrane potential and the voltage-step-induced currents (Figures 4-5). In comparison, 9-phenanthrol-induced hyperpolarization and attenuated the voltage-step-induced currents. 3,15 CBA, hence, did not mimic the inhibitory effects of 9-phenanthrol in guinea pig UBSM.
Several causes may underlie differences in the effects of 9-phenanthrol and CBA. First, 9-phenanthrol mediated effects could be due to non-TRPM4 channel pharmacology. 9-Phenanthrol is not very selective for TRPM4 channels as this compound also acts on TMEM16A/Ano1 Cl − , Ca V , Na V , K V , and IK channels and induces cytotoxicity. [21][22][23][24][25] In UBSM cells, 9-phenanthrol also increased cell capacitance, an effect independent of TRPM4 channel involvement. 15  Third, TRPM4 channels in UBSM exist in a configuration state regardless of species that exhibit a unique pharmacological profile: sensitivity to 9-phenanthrol, insensitivity to CBA and FFA, and only partial inhibition by glibenclamide, another non-selective TRPM4 channel blocker. 15 Among these explanations, we favor the first that the effects of 9-phenanthrol in UBSM are due to a non-TRPM4 channel interaction.
Other confounding factors in our electrophysiological experiments are the temperature-dependency of TRPM4 channels and dependency on intracellular Ca 2+ . 39 Half voltage activation constant for TRPM4 currents shifted ~ −50 mV when changed from 20 to 31°C, and the channel opening required high Ca 2+ (>10-100 μM). 39 Our electrophysiological recordings were conducted at room temperature and under condition where intracellular Ca 2+ levels could not be controlled experimentally. We acknowledge these limitations. TRPM4 currents, however, have been recorded in cerebral vascular smooth muscle cells using the same experimental approach as utilized here. 40 (Figures 1-3). Of note, for 20 mM KCl-induced contractions, we observed an enhancement in the frequency for the highest CBA concentrations, most likely due to non-selectivity.
As CBA has been recently shown to inhibit transient outward K + current and late Na + current in ventricular myocytes, 36 this compound may lack optimal selectivity. Given the issues with the currently available TRPM4 channel pharmacological tools, determining the roles of TRPM4 channel function in UBSM will require additional approaches. The utility  * of genetic animal models, whether global or inducible conditional, will be extremely useful as has been the case for elucidating the roles of TRPM4 channels in the heart, taste buds, brain, and pancreas. 3 8 ,42-4 6 Unfortunately, the specific TRPM4 knockout mouse or rat strains have not yet been made available to us.
We look forward to these prospective studies providing further insights.
As TRPM4 channels are robustly expressed in UBSM cells and tissues of humans, rats, guinea pigs, and mice, 3-5,7,12 a question arises as to their overall physiological role in the urinary bladder. Our current study provides evidence that they may not directly regulate UBSM excitability or contractility. For non-smooth muscle cells, TRPM4 channels have been implicated in the regulation of hypertrophy, cell growth, proliferation, migration, and cell differentiation promoting fibrosis. 45,[47][48][49] Additionally, TRPM4 channels form interacting complexes with TRPC3 and SUR1 subunits as well as cytoskeletal adhesion proteins affecting their properties, 50-52 although in healthy UBSM cells TRPM4-SUR1 complexes are unlikely to be functional. 15 TRPM4 channels may play one of these roles in UBSM cells. A recent report found a time-dependent upregulation of TRPM4 channel expression in the bladder of a spinal cord injury mouse model suggesting a contribution of TRPM4 channels to post-injury healing. 7 TRPM4 channel protein expression also changed during maturation from youth to adulthood in guinea pigs for both intracellular and cell surface expression perhaps related to the putative non-excitatory, non-contractile regulatory role. 13 Additional mechanistic studies are, therefore, needed to unravel the functional role of TRPM4 channels in UBSM.
In summary, the novel TRPM4 channel inhibitor CBA did not mimic the inhibitory effects of the classical and non-selective inhibitor 9-phenanthrol on UBSM electrical and mechanical activities.
Two other TRPM4 channel modulators FFA and BTP2 did not affect UBSM cation currents as expected for the TRPM4 channel engagement. Our study, hence, suggests that TRPM4 channels, although expressed in UBSM, play an additional role (e.g., in cell proliferation or under pathophysiological conditions) rather than regulation of excitability and contractility.

D I SCLOS U R E
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the findings of this study are available from the corresponding authors upon request.

E TH I C S S TATEM ENT
Experiments were conducted in accordance with the Animal Use Protocols Nos. 17-075 and 20-019 reviewed and approved by the University of Tennessee Health Science Center (Memphis, TN).